CN106906248A - A kind of method of the propane diols of utilization recombinant microorganism fermenting and producing 1,3 - Google Patents
A kind of method of the propane diols of utilization recombinant microorganism fermenting and producing 1,3 Download PDFInfo
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- CN106906248A CN106906248A CN201710194075.1A CN201710194075A CN106906248A CN 106906248 A CN106906248 A CN 106906248A CN 201710194075 A CN201710194075 A CN 201710194075A CN 106906248 A CN106906248 A CN 106906248A
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- dha
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- glycerol
- dihydroxyacetone
- dehydrogenase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
Abstract
The invention provides a kind of method of the propane diols of utilization recombinant microorganism fermenting and producing 1,3.The present invention strengthens phosphoric acid dihydroxyacetone (DHA) dephosphorylation generation dihydroxyacetone (DHA) by the endogenous phosphoric acid dihydroxyacetone (DHA) phosphatase gene hdpA of the overexpression in Corynebacterium glutamicum, dihydroxyacetone (DHA) is changed into glycerine by the glycerol dehydrogenase for introducing external source, glycerine ultimately generates 1,3 propane diols in the presence of the glycerol dehydratase and its activity factor and alcohol dehydrogenase of external source.Corynebacterium glutamicum can be fermented using different cheap raw materials, cheap corn pulp can also be used to substitute expensive dusty yeast as nutritional ingredient, the cost of raw material can be further reduced, while solving biological safety and bacterial strain to substrate and the problem of resistance of product.The thalline in fermentation process can be as product, in the middle of feed addictive simultaneously.The inventive method accessory substance is few, can further simplify the separation process of 1,3 propane diols.
Description
Technical field
The invention belongs to genetic engineering and technical field of biological fermentation, specifically, can by single recombinant microorganism
The method that sugar fermentation high-performance bio is converted into 1,3- propane diols.
Background technology
1,3-PD is a kind of important industrial chemicals, can be applied to ink, printing and dyeing, coating, profit as organic solvent
The industries such as lubrication prescription, antifreeze, its topmost purposes is the monomer synthesized as polyester and polyurethane, particularly and terephthaldehyde
Acid polymerization generation polytrimethylene terephthalate (PTT).PTT and PET (polyethylene terephthalate), PBT are (poly- to benzene two
Formic acid butanediol ester) compared to more excellent performance, such as more preferable stain resistance, toughness and resilience and ultraviolet-resistent property
Can wait, additionally have the advantages that wear-resisting, water imbibition is low, low electrostatic.Therefore PTT is considered as the upgrading products of PET, with wide
Wealthy market prospects.
At present, the production method of 1,3-PD mainly includes chemical method and bioanalysis.Chemical method is typically with epoxy third
Alkane or propylene are that raw material synthesizes 1,3-PD by complicated catalytic process.The shortcoming of chemical synthesis is many accessory substances,
Poor selectivity, operating condition needs HTHP, and equipment investment is huge, and raw material is non-renewable resources.Therefore, chemical method production 1,
The processing technology routine of ammediol is substantially superseded at present.
Bioanalysis production 1,3-PD mainly includes two technology paths at present:First, it is that raw material is utilized naturally with glycerine
Micro-organisms 1,3-PD;2nd, 1,3-PD is produced using recombinant microorganism by raw material of glucose.It is specific to introduce such as
Under.
One kind is to produce 1,3-PD using natural microbial by raw material of glycerine.There is natural micro- life in nature
Thing, such as Klebsiella pneumoniae (Klebsiella pneumoniae), butyric acid clostruidium (Clostridium
) and C. freundii (Citrobacter freundii) can will be sweet under conditions of anaerobism or micro- oxygen butyricum
Oil is converted into 1,3-PD, and its metabolic pathway mainly includes two-step reaction, the first step:Glycerine is in the presence of glycerol dehydratase
Dehydration generation 3-HPA, second step:3-HPA is reduced into 1,3- the third two under the catalysis of 1,3-PD dehydrogenase
Alcohol.Wherein glycerol dehydratase is made up of 3 subunits, and its encoding gene is dhaBCE (or dhaB123).Glycerol dehydratase is being urged
Can be inactivated automatically, it is necessary to be activated again in the presence of activity factor during changing dehydrating glycerin.Dehydrating glycerin enzyme activition
The encoding gene of the factor is dhaG and dhaH.Also there is 1,2-PD dehydratase and its activity factor in nature simultaneously can be with
The dehydration of catalyzing glycerol or 1,2-PD, its encoding gene is pduCDEGH.1,3-PD dehydrogenase is a NADH
The alcohol dehydrogenase of dependence, its encoding gene is dhaT.YqhD gene codes in some other alcohol dehydrogenase such as Escherichia coli
The alcohol dehydrogenase that NADPH is relied on can also be catalyzed 3-HPA also original production 1,3-PD.Glycerine is in reduction generation 1,3-
The process of propane diols is, it is necessary to consume reducing power NADH or NADPH, these reducing powers are to be oxidized to other accessory substances by glycerine
Such as acetic acid, lactic acid, the generation such as 2,3-butanediol.
At present, the industrial technique using glycerol production 1,3-PD is mainly using Klebsiella pneumoniae (as in
State patent CN 200810105722.8).The major defect of this process route is:First, Klebsiella pneumoniae is condition cause
Germ, biological safety needs strict control in its production process;2nd, a large amount of accessory substances such as acetic acid, lactic acid, succinic acid, 2,3-
The synthesis of butanediol so that extraction process becomes sufficiently complex after whole;3rd, the yield of 1,3-PD is low, usual 1,3- third
Glycol is less than 40% to the quality yield of glycerine.
There is a kind of side for being to be produced using recombination bacillus coli as raw material with glucose 1,3-PD in the prior art
Method.Nature in the absence of can directly transforming glucose generation 1,3-PD microorganism.E.I.Du Pont Company is by large intestine bar
Glycerine route of synthesis (glyceraldehyde-3 phosphate dehydrogenase and glyceraldehyde-3 phosphate enzyme) of the heterogenous expression from saccharomyces cerevisiae and come from bacterium
The glycerol dehydratase and its activity factor of Klebsiella pneumoniae and the alcohol dehydrogenase of the NADPH dependences using Escherichia coli itself
Enzyme YqhD, realizes step conversion (CN 200380104657.2) from glucose to 1,3-PD.This process route
Have the disadvantage that Escherichia coli are poor biological safeties, the fermented and cultured of Escherichia coli needs to use the raw material dusty yeast of costliness, because
This technology is unfavorable for that industrialized scale metaplasia is produced.
The content of the invention
It is an object of the invention to provide a kind of biological safety it is high, yield is high, easy to operate and with low cost using weight
The method of group 1,3-propanediol produced by microbial fermentation, to overcome the deficiencies in the prior art.
1,3- propane diols route of synthesis proposed by the present invention is as shown in Figure 1.The fermentable sugars such as glucose or sucrose passes through
Glycolytic pathway produces phosphoric acid dihydroxyacetone (DHA), and the latter generates dihydroxy third under the catalysis of phosphoric acid dihydroxyacetone (DHA) phosphate
Ketone, the latter further synthesizes into glycerine under the catalysis of glycerol dehydrogenase.Effect of the glycerine in glycerol dehydratase and alcohol dehydrogenase
Under ultimately generate 1,3- propane diols.The approach is different from the 1,3-PD synthetic method that E.I.Du Pont Company uses, and the main distinction is
E.I.Du Pont Company is by expressing glyceraldehyde-3 phosphate dehydrogenase and glyceraldehyde-3 phosphate lipase come synthetic mesophase glycerol production, and the present invention is carried
The approach for going out can realize the synthesis from glucose to 1,3- propane diols in different microorganisms.
A kind of method of utilization recombinant microorganism producing 1,3-propanediol through fermentation that the present invention is provided, comprises the following steps:
(1) building being capable of overexpression phosphoric acid dihydroxyacetone (DHA) phosphate, glycerol dehydrogenase, glycerol dehydratase and its activation
The recombinant microorganism of the factor, alcohol dehydrogenase;
(2) aerobic fermentation is carried out by substrate of the raw material containing fermentable sugars.
In the method for the present invention, the construction method of the recombinant microorganism is:1) build and be able to overexpression di(2-ethylhexyl)phosphate
The recombinant bacterium or recombinant plasmid of hydroxypropanone- phosphate and glycerol dehydrogenase;2) build structure be able to overexpression glycerine take off
The recombinant bacterium or recombinant plasmid of water enzyme and its activity factor and alcohol dehydrogenase;3) by step 1) obtain recombinant plasmid electricity be transformed into
Step 2) in the recombinant bacterium that obtains, or by step 2) the recombinant bacterium electricity that obtains is transformed into step 1) in the recombinant bacterium that obtains, screening
Obtaining being capable of overexpression phosphoric acid dihydroxyacetone (DHA) phosphate, glycerol dehydrogenase, glycerol dehydratase and its activity factor, alcohol dehydrogenase
The recombinant microorganism of enzyme.
Described microorganism is Corynebacterium glutamicum, Escherichia coli, bacillus subtilis, saccharomyces cerevisiae.
The present invention builds the route of synthesis of 1,3- propane diols by the use of Corynebacterium glutamicum as host microorganism.Glutamic acid
Bar bacterium is a kind of microorganism of aliment security level, is widely used in the production of amino acid, such as the life of glutamic acid and lysine
Produce.Using Corynebacterium glutamicum biological safety is solved the problems, such as come fermenting and producing 1,3- propane diols.
Phosphoric acid dihydroxyacetone (DHA) phosphate of the present invention derives from Corynebacterium glutamicum, its nucleotide sequence such as SEQ
Shown in ID NO.1;The glycerol dehydrogenase derives from Escherichia coli, and its nucleotide sequence is as shown in SEQ ID NO.2.
Further, phosphoric acid dihydroxyacetone (DHA) phosphate hdpA preparation methods of the invention are:With Corynebacterium glutamicum
The genome of ATCC 13032 is template, with hdpA-F
And hdpA-R (cagctatgaccatgattacgATAAGGCTGATTAGCGGGAAAATTTCG)
(CCAGTCGTGTCTAGTCAGTGAACTGCTGCTCATCT) for primer enters performing PCR, hdpA gene (hdpA gene orders are obtained
Such as SEQ ID NO.1) about 0.9kb goes forward side by side performing PCR product purification.
Glycerol dehydrogenase gldA of the invention with the genome of Escherichia coli MG1655 as template, with gldA-F
And gldA-R (CACTGACTAGACACGACTGGAATGCCGC)
(atccccgggtaccgagctcgttaTTCCCACTCTTGCAGGAAACGC) for primer enters performing PCR, gldA genes are obtained
(gldA gene orders such as SEQ ID NO.2) about 1.2kb goes forward side by side performing PCR product purification.
Preferably, the glycerol dehydratase and its activity factor, its nucleotide sequence as shown in SEQ ID NO.3, glycerine
Dehydratase and its activity factor derive from Klebsiella pneumoniae.
In an embodiment of the present invention, glycerol dehydratase and its activity factor are with Klebsiella pneumoniae HR526
Genome is template, with pdu-F (CGCCCGCtaaAAGGAGATATACCATGAGATCGAAAAGATTTGAAG) and pdu-R
(tgcctgcaggtcgactctagTTAAGCATGGCG) for primer enters performing PCR, acquisition include glycerol dehydratase and its activate because
The pduCDEGH of son manipulates sub-piece (sequence such as SEQ ID NO.3) performing PCR product purification of going forward side by side.
It will be appreciated by those skilled in the art that in addition to expressing the glycerol dehydratase from Klebsiella pneumoniae, table
Identical function can be equally realized up to other glycerol dehydratases originated.
In the method for utilization recombinant microorganism producing 1,3-propanediol through fermentation of the invention, the alcohol dehydrogenase comes from large intestine
Bacillus or Klebsiella Pneumoniae.
In one embodiment of the invention, the preparation method of alcohol dehydrogenase yqhD is, with the base of Escherichia coli MG1655
Because group is template, with primer yqhD-F (ctatgacatgattacgaattAAGGAGATATACCatgAACAACTTTAATCTGCA
C) and yqhD-R (ATATCTCCTTttaGCGGGCGGCTTCG) enters performing PCR for primer, alcohol dehydrogenase gene yqhD fragments are obtained
(sequence is as shown in SEQ ID NO.3) is simultaneously purified.
In the method for utilization recombinant microorganism producing 1,3-propanediol through fermentation of the invention, step (2) is described to be contained and can ferment
The raw material of sugar is molasses, sucrose, glucose, starch hydrolyzate, corn pulp, xylose, ligno-cellulose hydrolysate or glycerine.
The fermentation condition of step (2) is, 28-35 DEG C, pH value 5-8, oxygen dissolving value 10%.Can control to ferment by Feeding ammonia water
The pH value of system.
Preferably, fermentation temperature is 30-32 DEG C, and pH value is 6-7.
Fermentation medium includes sugar, KH2PO4, MgSO4, MnSO4, FeSO4, corn pulp, (NH4)2SO4, thiamine hydrochloride, dimension
Raw element B12.
The method of utilization recombinant microorganism fermenting and producing 1,3- propane diols of the invention also includes stream plus carbon in fermentation process
Contain thiamine and vitamin B12 in source, and fermentation medium.
Application of the above method in 1,3- propanediols are improved belongs to protection scope of the present invention.
The present invention is by the endogenous phosphoric acid dihydroxyacetone (DHA) phosphatase gene hdpA of the overexpression in Corynebacterium glutamicum
To strengthen phosphoric acid dihydroxyacetone (DHA) dephosphorylation generation dihydroxyacetone (DHA), the glycerol dehydrogenase for introducing external source converts dihydroxyacetone (DHA)
Into glycerine, glycerine ultimately generates 1,3- the third two in the presence of the glycerol dehydratase and its activity factor and alcohol dehydrogenase of external source
Alcohol.Corynebacterium glutamicum can be fermented using different cheap raw materials, can also use cheap corn pulp as nutrition
Composition substitutes expensive dusty yeast, the cost of raw material can be further reduced, while solving biological safety and bacterial strain pair
The problem of resistance of substrate and product.Thalline in fermentation process can be as product, in the middle of feed addictive.The present invention
Method is simple to operate, with low cost, and 1,3-PD yield is high, and accessory substance is few, is conducive to further simplifying 1,3-PD
Separation process.
Brief description of the drawings
Fig. 1 is the flow chart that the present invention produces 1,3- propane diols methods using recombinant microorganism glucose fermentation.
Specific embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Without departing substantially from spirit of the invention
In the case of essence, the modification or replacement made to the inventive method, step or condition belong to the scope of the present invention.
If not specializing, chemical reagent used is conventional commercial reagent in embodiment, skill used in embodiment
The conventional meanses that art means are well known to those skilled in the art.
The overexpression phosphoric acid dihydroxyacetone (DHA) phosphatase gene of embodiment 1 and the glycerol dehydrogenase base from Escherichia coli
Because of gldA
Genome with Corynebacterium glutamicum ATCC 13032 as template, with primer hdpA-F
And hdpA-R (cagctatgaccatgattacgATAAGGCTGATTAGCGGGAAAATTTCG)
(CCAGTCGTGTCTAGTCAGTGAACTGCTGCTCATCT) for primer enters performing PCR, hdpA gene (hdpA gene orders are obtained
Such as SEQ ID NO.1) about 0.9kb goes forward side by side performing PCR product purification.
Genome with Escherichia coli MG1655 as template, with primer gldA-F
And gldA-R (CACTGACTAGACACGACTGGAATGCCGC)
(atccccgggtaccgagctcgttaTTCCCACTCTTGCAGGAAACGC) for primer enters performing PCR, gldA genes are obtained
(gldA gene orders such as SEQ ID NO.2) about 1.2kb goes forward side by side performing PCR product purification.
By E. coli-C. glutamicum shuttle plasmid pEC-K18mob2 (Journal of Biotechnology
104 (2003) 287-299) carry out digestion with EcoRI, using Gibson Assembly kits (NEB) by hpdA fragments and
The step of gldA fragments one is connected on pEC-K18mob2, and the recombinant plasmid of acquisition is named as pEC-hdpA-gldA.
PEC-hdpA-gldA is transferred to Corynebacterium glutamicum ATCC by electricity using electroporation apparatus (Bole)
In 13032, electric shock condition is voltage 2.5KV, the Ω of resistance 600, the μ F of electric capacity 25 (electric shock cup width is 2mm).Containing 25mg/L's
Screened on kanamycins LB flat boards and obtain recombinant bacterium, be named as C.glutamicum/pEC-hdpA-gldA.
The overexpression of embodiment 2 from Klebsiella pneumoniae glycerol dehydratase and its activity factor pduCDEGH and
Alcohol dehydrogenase gene yqhD from Escherichia coli
Genome with Klebsiella pneumoniae HR526 as template, with primer pdu-F
And pdu-R (CGCCCGCtaaAAGGAGATATACCATGAGATCGAAAAGATTTGAAG)
(tgcctgcaggtcgactctagTTAAGCATGGCG) for primer enters performing PCR, acquisition include glycerol dehydratase and its activate because
The pduCDEGH of son manipulates sub-piece (sequence is as shown in SEQ ID NO.3) performing PCR product purification of going forward side by side.
Genome with Escherichia coli MG1655 as template, with primer yqhD-F
And yqhD-R (ctatgacatgattacgaattAAGGAGATATACCatgAACAACTTTAATCTGCAC)
(ATATCTCCTTttaGCGGGCGGCTTCG) for primer enters performing PCR, alcohol dehydrogenase gene yqhD fragments (sequence such as SEQ is obtained
Shown in ID NO.4) and purified.
By E. coli-C. glutamicum shuttle plasmid pXMJ19【Jakoby, M.J., Ngouto-Nkili are disclosed in,
C.E.and Burkovski,A.Construction and application of new Corynebacterium
glutamicum vectors.JOURNAL BioTechniques13,437-441(1999)】Digestion is carried out with XbaI, is utilized
PduCDEGH is manipulated Gibson Assembly kits (NEB) into sub-piece and the step of yqhD fragments one is connected on pXMJ19, obtains
The recombinant plasmid for obtaining is named as pXMJ-yqhD-pdu.
PXMJ-yqhD-pdu is transferred to Corynebacterium glutamicum by electricity using electroporation apparatus (Bole)
In C.glutamicum/pEC-hdpA-gldA, electric shock condition is as described in Example 1.In chloramphenicol and 25mg/L containing 5mg/L
Kanamycins LB flat boards on screen obtain recombinant bacterium, be named as C.glutamicum/pEC-hdpA-gldA/pXMJ-yqhD-
pdu。
Embodiment 3 recombinates the fermented and cultured of Corynebacterium glutamicum
By Corynebacterium glutamicum wild strain ATCC13032 and recombinant bacterial strain C.glutamicum/ obtained in embodiment 1
PEC-hdpA-gldA, recombinant bacterium C.glutamicum/pEC-hdpA-gldA/pXMJ-yqhD-pdu is in LB obtained in embodiment 3
Plate overnight culture.The 250ml band plate washer shaking flasks containing 30ml seed culture mediums are inoculated into from single bacterium colony on the fresh plate
In, 32 DEG C, 200rpm is cultivated 12 hours.
The formula of seed culture medium includes (g/L):Glucose 25, (NH4)2SO4 5.0,K2HPO4 1.5,MgSO4 1.0,
MnSO4 0.005,FeSO40.005, corn pulp 30, kanamycins 0.025.
Seed liquor is inoculated into the middle of 2L fermentation mediums with 10% inoculum concentration, fermentation is controlled using the fermentation tank of 5L
Temperature is 32 DEG C, and throughput is 1vvm, and adjustment rotating speed causes that dissolved oxygen level is maintained at more than 10%, and pH is controlled by Feeding ammonia water
Value stabilization is 7.0 or so.
Fermentative medium formula includes (g/L):Glucose 100, (NH4)2SO4 30.0,K2HPO42.5,MgSO4 1.0,
MnSO4 0.010,FeSO40.010, corn pulp 15, biotin 0.0005, thiamine hydrochloride 0.005, vitamin B12 0.005.
Fermentation 48 hours, using high performance liquid chromatography detection tunning wild strain ATCC13032 do not produce glycerine and
1,3-PD, C.glutamicum/pEC-hdpA-gldA produces the glycerine of 52g/L but does not produce 1,3-PD.
C.glutamicum/pEC-hdpA-gldA/pXMJ-yqhD-pdu produces the 1,3-PD of 32g/L, and yield reaches every gram of grape
Sugar produces 0.32g 1,3- propane diols.Without the accessory substances such as formic acid, succinic acid, 2,3-butanediol are produced in fermentation, with respect to other works
For skill, it is easy to follow-up purifying technique.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
SEQUENCE LISTING
<110>Tsing-Hua University
<120>A kind of method of utilization recombinant microorganism fermenting and producing 1,3- propane diols
<130> KHP171111659.2
<160> 12
<170> PatentIn version 3.5
<210> 1
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atgacagtga acatttcata tctgaccgac atggacggcg tcctcatcaa agagggcgag 60
ataattccgg gtgcagatcg ttttcttcag tctctcaccg ataacaatgt ggagtttatg 120
gttttgacca acaactccat tttcaccccg agggatcttt ctgcacgtct taagacttcc 180
ggtttggaaa tcccgccgga gcgtatttgg acttctgcaa ccgccactgc tcacttcctg 240
aaatcccagg tcaaggaggg cacagcctat gttgttggcg agtccggtct gaccactgcg 300
ttgcataccg cgggttggat tttgacggat gcaaatcctg agtttgttgt cctgggcgaa 360
acccgcacat attccttcga ggcaatcact actgcgataa atctgatttt gggtggcgct 420
cgctttattt gcaccaaccc ggatgtcact ggaccttcac caagtggcat tttgcctgct 480
actggctctg tcgccgcact tattaccgca gctactggcg ctgagcctta ttacatcggc 540
aagccaaacc ctgtgatgat gcgcagtgcg ctgaacacca tcggggcgca ttccgagcac 600
actgtcatga tcggcgaccg catggacacc gacgtgaaat ctggtttgga agccggcctg 660
agcaccgtgc tggttcgaag cggaatttcc gacgacgccg agatccgccg ctaccccttc 720
cgcccaactc acgtgatcaa ttccatcgcc gatcttgccg attgctggga cgatcctttc 780
ggtgacggtg catttcacgt accagatgag cagcagttca ctgactag 828
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ttaggttttg ctcaatccac tgtcgagaaa agctttaaag atgctggact ggtagtagaa 180
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gagactgcgc agtgtggcgc aattctcggt atcggtggcg gaaaaaccct cgatactgcc 300
aaagcactgg cacatttcat gggtgttccg gtagcgatcg caccgactat cgcctctacc 360
gatgcaccgt gcagcgcatt gtctgttatc tacaccgatg agggtgagtt tgaccgctat 420
ctgctgttgc caaataaccc gaatatggtc attgtcgaca ccaaaatcgt cgctggcgca 480
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gcctgctctc gtagcggcgc gaccaccatg gcgggcggca agtgcaccca ggctgcgctg 600
gcactggctg aactgtgcta caacaccctg ctggaagaag gcgaaaaagc gatgcttgct 660
gccgaacagc atgtagtgac tccggcgctg gagcgcgtga ttgaagcgaa cacctatttg 720
agcggtgttg gttttgaaag tggtggtctg gctgcggcgc acgcagtgca taacggcctg 780
accgctatcc cggacgcgca tcactattat cacggtgaaa aagtggcatt cggtacgctg 840
acgcagctgg ttctggaaaa tgcgccggtg gaggaaatcg aaaccgtagc tgcccttagc 900
catgcggtag gtttgccaat aactctcgct caactggata ttaaagaaga tgtcccggcg 960
aaaatgcgaa ttgtggcaga agcggcatgt gcagaaggtg aaaccattca caacatgcct 1020
ggcggcgcga cgccagatca ggtttacgcc gctctgctgg tagccgacca gtacggtcag 1080
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aaggagtgga ttgaagaggg ctttatcgcg atggaaagcc ctaacgatcc caaaccttct 120
atccgcatcg tcaacggcgc ggtgaccgaa ctcgacggta aaccggttga gcagttcgac 180
ctgattgacc actttatcgc gcgctacggc attaatctcg cccgggccga agaagtgatg 240
gccatggatt cggttaagct cgccaacatg ctctgcgacc cgaacgttaa acgcagcgac 300
atcgtgccgc tcactaccgc gatgaccccg gcgaaaatcg tggaagtggt gtcgcatatg 360
aacgtggtcg agatgatgat ggcgatgcaa aaaatgcgcg cccgccgcac gccgtcccag 420
caggcgcatg tcactaatat caaagataat ccggtacaga ttgccgccga cgccgctgaa 480
ggcgcatggc gcggctttga cgaacaggag accaccgtcg ccgtggcgcg ctacgcgccg 540
ttcaacgcca tcgccctgct ggtgggttca caggttggcc gccccggcgt cctcacccag 600
tgttcgctgg aagaagccac cgagctgaaa ctgggcatgc tgggccacac ctgctatgcc 660
gaaaccattt cggtatacgg tacggaaccg gtgtttaccg atggcgatga cactccatgg 720
tcgaaaggct tcctcgcctc ctcctacgcc tcgcgcggcc tgaaaatgcg ctttacctcc 780
ggttccggtt ctgaagtaca gatgggctat gccgaaggca aatcgatgct ttatctcgaa 840
gcgcgctgca tctacatcac caaagccgcc ggggtgcaag gcctgcagaa tggctccgtc 900
agctgtatcg gcgtaccgtc cgccgtgccg tccgggatcc gcgccgtact ggcggaaaac 960
ctgatctgct cagcgctgga tctggagtgc gcctccagca acgatcaaac ctttacccac 1020
tcggatatgc ggcgtaccgc gcgtctgctg atgcagttcc tgccaggcac cgacttcatc 1080
tcctccggtt actcggcggt gccgaactac gacaacatgt tcgccggttc caacgaagat 1140
gccgaagact tcgatgacta caacgtgatc cagcgcgacc tgaaggtcga tggcggcctg 1200
cggccggtgc gtgaagagga cgtgatcgcc attcgcaaca aagccgcccg cgcgctgcag 1260
gcggtatttg ccggcatggg tttgccgcct attacggatg aagaagtaga agccgccacc 1320
tacgcccacg gttcaaaaga tatgcctgag cgcaatatcg tcgaggacat caagtttgct 1380
caggagatca tcaacaagaa ccgcaacggc ctggaagtgg tgaaagccct ggcgaaaggc 1440
ggcttccccg atgtcgccca ggacatgctc aatattcaga aagccaagct caccggcgac 1500
tacctgcata cctccgccat cattgttggc gagggccagg tgctctcggc cgtgaatgac 1560
gtgaacgatt atgccggtcc ggcaacaggc taccgcctgc aaggcgagcg ctgggaagag 1620
attaaaaata tcccgggcgc gctcgatccc aatgaacttg gctaaggggt gaaaaatgga 1680
aattaacgaa acgctgctgc gccagattat cgaagaggtg ctgtcggaga tgaaatcagg 1740
cgcagataag ccggtctcct ttagcgcgcc tgcggcttct gtcgcctctg ccgcaccggt 1800
cgccgttgcg cctgtgtccg gcgacagctt cctgacggaa atcggcgaag ccaaacccgg 1860
cacgcagcag gatgaagtca ttattgccgt cgggccagcg tttggtctgg cgcaaaccgc 1920
caatatcgtc ggcattccgc ataaaaatat tctgcgcgaa gtgatcgccg gcattgagga 1980
agaaggcatc aaagcccggg tgatccgctg ctttaagtct tctgacgtcg ccttcgtggc 2040
agtggaaggc aaccgcctga gcggctccgg catctcgatc ggtattcagt cgaaaggcac 2100
caccgtcatc caccagcgcg gcctgccgcc gctttccaat ctggaactct tcccgcaggc 2160
gccgctgctg acgctggaaa cctaccgtca gattggcaaa aacgccgcgc gctacgccaa 2220
acgcgagtcg ccgcagccgg tgccgacgct taacgatcag atggctcgtc ccaaatacca 2280
ggcgaagtcg gccattttgc acattaaaga gaccaaatac gtggtgacgg gcaaaaaccc 2340
gcaggaactg cgcgtggcgc tttaacaaag gatatcccga tgaataccga cgcaattgaa 2400
tccatggtac gcgacgtgct gagccggatg aacagcctac aggacgggat aacgcccgcg 2460
ccagccgcgc cgacaaacga caccgttcgc cagccaaaag ttagcgacta cccgttagcg 2520
acccgccatc cggagtgggt caaaaccgct accaataaaa cgctcgatga cctgacgctg 2580
gagaacgtat taagcgatcg cgttacggcg caggacatgc gcatcactcc ggaaacgctg 2640
cgtatgcagg cggcgatcgc ccaggatgcc ggacgcgatc ggctggcgat gaactttgag 2700
cgggccgcag agctcaccgc ggttcccgac gaccgaatcc ttgagatcta caacgccctg 2760
cgcccatacc gttccaccca ggcggagcta ctggcgatcg ctgatgacct cgagcatcgc 2820
taccaggcac gactctgtgc cgcctttgtt cgggaagcgg ccgggctgta catcgagcgt 2880
aagaagctga aaggcgacga ttaacagggg gtaagcatgc gctatatcgc tggcattgat 2940
attggcaact cctcgacaga agtcgccctg gcgacggtcg atgacgcagg tgtgctgaac 3000
actcgccaca gcgcgttggc tgaaaccacg ggtataaaag gcacattacg aaatgtgttc 3060
ggtatccagg aggcgctaac gcaggcggca aaagcggccg gcattcagct cagcgatatt 3120
tcgcttattc gcattaacga agccacgccg gtcattggcg atgtggcgat ggaaaccatc 3180
acggaaacca tcatcaccga gtccaccatg atcggccata acccgaagac acccggcggc 3240
gtcggactgg gggtcggcat caccatcaca ccagaggcgc tgctgtcctg ctccgcggac 3300
actccctata ttctggtggt ctcctcggcc tttgactttg ccgatgtcgc cgcgatggtc 3360
aatgcggcaa cggcagcggg ctatcagata accggcatta ttttgcagca ggatgacggc 3420
gtgctggtca ataaccggct acagcaaccg ctaccggtga tcgacgaagt tcagcatatc 3480
gaccggattc cacttggcat gctggcggcc gtcgaggtcg ctttacccgg taagatcatc 3540
gaaacgctct ccaaccccta cggtattgcg accgttttcg atctcaacgc cgaggagacc 3600
aaaaatatcg tgccaatggc gcgggcgctg attggcaacc gctcggccgt ggtggtgaaa 3660
accccctccg gcgacgtcaa ggcccgcgct attccggcag gtaatctgct gctcatcgct 3720
caagggcgca gcgtacaggt tgatgtggcc gccggggcgg aagccatcat gaaagcggtt 3780
gacggctgcg gcaaactgga caacgtcgcg ggagaagcgg gcaccaatat cggcggcatg 3840
ctagagcacg tgcgccagac catggcggag cttaccaata agccagctca ggagatccgc 3900
attcaggatc tgctggccgt tgatacggcg gtgccagtca gcgtgaccgg cggtcttgcg 3960
ggggagttct cgctggagca ggcggtgggt atcgcctcga tggtcaagtc ggatcgcctg 4020
cagatggccc tcatcgcccg tgaaattgag cacaaactgc agattgcggt tcaggtgggc 4080
ggcgccgaag cggaggcggc cattcttggg gcgctcacca ctcccggcac cacgcgcccg 4140
ctggcgatcc tcgatctggg cgccgggtcg accgacgcct ccattatcaa tgcgcaggga 4200
gagatcagcg ccactcacct ggccggcgcc ggcgatatgg tcacgatgat catcgcccgc 4260
gagctggggc ttgaggaccg ctacctggcg gaagagatca aaaaatatcc gctggctaaa 4320
gtcgaaagcc tgtttcatct gcgtcatgaa gacggcagcg tccagttttt tccgtcggcc 4380
ttaccaccga cggtatttgc ccgcgtctgc gtggtgaaac cggatgaact ggttcccctg 4440
cccggcgatc tgccgctgga gaaagtgcgc gccattcgcc gtagcgccaa atcacgcgtc 4500
tttatcacca acgccctgcg agcgttacgc caggtgagcc ctaccggcaa cattcgcgac 4560
atcccgttcg tggtgctggt gggcggctcg tccctcgatt tcgagatccc ccagctggtc 4620
accgacgcgc tggcgcacta ccggctggtt gccgggcgcg gcaacatccg cggctgtgaa 4680
ggcccacgca atgcggtcgc cagcggatta ctcctttcct ggcaaaaagg aggcacacat 4740
ggagagtagc gtagtcgccc ccgccatcgt cattgccgtc actgacgaat gcagcgaaca 4800
gtggcgcgat gtcctgctgg gcattgaaga ggaaggcatt ccttttgttc tgcagccgca 4860
gaccggcggc gatcttatcc atcacgcctg gcaggcggcg cagcgttcgc cgctgcaggt 4920
aggcatcgcc tgcgaccggg aacggctcat cgtgcactac aaaaatttac ccgcatcaac 4980
tccgttgttt tcgctgatgt atcaccagaa caggctggcc cggcgaaaca ctggcaacaa 5040
tgcggctcgt ctcgtcaaag ggatcccatt tcgggatcgc catgcttaa 5089
<210> 4
<211> 1164
<212> DNA
<213> yqhD
<400> 4
atgaacaact ttaatctgca caccccaacc cgcattctgt ttggtaaagg cgcaatcgct 60
ggtttacgcg aacaaattcc tcacgatgct cgcgtattga ttacctacgg cggcggcagc 120
gtgaaaaaaa ccggcgttct cgatcaagtt ctggatgccc tgaaaggcat ggacgtgctg 180
gaatttggcg gtattgagcc aaacccggct tatgaaacgc tgatgaacgc cgtgaaactg 240
gttcgcgaac agaaagtgac tttcctgctg gcggttggcg gcggttctgt actggacggc 300
accaaattta tcgccgcagc ggctaactat ccggaaaata tcgatccgtg gcacattctg 360
caaacgggcg gtaaagagat taaaagcgcc atcccgatgg gctgtgtgct gacgctgcca 420
gcaaccggtt cagaatccaa cgcaggcgcg gtgatctccc gtaaaaccac aggcgacaag 480
caggcgttcc attctgccca tgttcagccg gtatttgccg tgctcgatcc ggtttatacc 540
tacaccctgc cgccgcgtca ggtggctaac ggcgtagtgg acgcctttgt acacaccgtg 600
gaacagtatg ttaccaaacc ggttgatgcc aaaattcagg accgtttcgc agaaggcatt 660
ttgctgacgc taatcgaaga tggtccgaaa gccctgaaag agccagaaaa ctacgatgtg 720
cgcgccaacg tcatgtgggc ggcgactcag gcgctgaacg gtttgattgg cgctggcgta 780
ccgcaggact gggcaacgca tatgctgggc cacgaactga ctgcgatgca cggtctggat 840
cacgcgcaaa cactggctat cgtcctgcct gcactgtgga atgaaaaacg cgataccaag 900
cgcgctaagc tgctgcaata tgctgaacgc gtctggaaca tcactgaagg ttccgatgat 960
gagcgtattg acgccgcgat tgccgcaacc cgcaatttct ttgagcaatt aggcgtgccg 1020
acccacctct ccgactacgg tctggacggc agctccatcc cggctttgct gaaaaaactg 1080
gaagagcacg gcatgaccca actgggcgaa aatcatgaca ttacgttgga tgtcagccgc 1140
cgtatatacg aagccgcccg ctaa 1164
<210> 5
<211> 47
<212> DNA
<213>Artificial sequence
<400> 5
cagctatgac catgattacg ataaggctga ttagcgggaa aatttcg 47
<210> 6
<211> 35
<212> DNA
<213>Artificial sequence
<400> 6
ccagtcgtgt ctagtcagtg aactgctgct catct 35
<210> 7
<211> 28
<212> DNA
<213>Artificial sequence
<400> 7
cactgactag acacgactgg aatgccgc 28
<210> 8
<211> 45
<212> DNA
<213>Artificial sequence
<400> 8
atccccgggt accgagctcg ttattcccac tcttgcagga aacgc 45
<210> 9
<211> 45
<212> DNA
<213>Artificial sequence
<400> 9
cgcccgctaa aaggagatat accatgagat cgaaaagatt tgaag 45
<210> 10
<211> 32
<212> DNA
<213>Artificial sequence
<400> 10
tgcctgcagg tcgactctag ttaagcatgg cg 32
<210> 11
<211> 54
<212> DNA
<213>Artificial sequence
<400> 11
ctatgacatg attacgaatt aaggagatat accatgaaca actttaatct gcac 54
<210> 12
<211> 26
<212> DNA
<213>Artificial sequence
<400> 12
atatctcctt ttagcgggcg gcttcg 26
Claims (10)
1. a kind of method of utilization recombinant microorganism producing 1,3-propanediol through fermentation, it is characterised in that comprise the following steps:
(1) build can overexpression phosphoric acid dihydroxyacetone (DHA) phosphate, glycerol dehydrogenase, glycerol dehydratase and its activation because
Son, the recombinant microorganism of alcohol dehydrogenase;
(2) aerobic fermentation is carried out by substrate of the raw material containing fermentable sugars.
2. the method for claim 1, it is characterised in that the construction method of the recombinant microorganism is:1) difference is built
It is capable of the recombinant bacterium or recombinant plasmid of overexpression phosphoric acid dihydroxyacetone (DHA) phosphate and glycerol dehydrogenase;2) build and be able to
The recombinant bacterium or recombinant plasmid of overexpression glycerol dehydratase and its activity factor and alcohol dehydrogenase;3) by step 1) obtain restructuring
Plasmid electricity is transformed into step 2) in the recombinant bacterium that obtains, or by step 2) the recombinant plasmid electricity that obtains is transformed into step 1) obtain
In recombinant bacterium, screening acquisition can overexpression phosphoric acid dihydroxyacetone (DHA) phosphate, glycerol dehydrogenase, glycerol dehydratase and its swash
The factor living, the recombinant microorganism of alcohol dehydrogenase.
3. method as claimed in claim 1 or 2, it is characterised in that described microorganism is Corynebacterium glutamicum, large intestine bar
Bacterium, bacillus subtilis, saccharomyces cerevisiae.
4. method as claimed in claim 2, it is characterised in that the phosphoric acid dihydroxyacetone (DHA) phosphate derives from glutamic acid
Bar bacterium, its nucleotide sequence is as shown in SEQ ID NO.1;The glycerol dehydrogenase derives from Escherichia coli, its nucleotides sequence
Row are as shown in SEQ ID NO.2.
5. method as claimed in claim 2, it is characterised in that the glycerol dehydratase and its activity factor, its nucleotides sequence
Row are as shown in SEQ ID NO.3.
6. method as claimed in claim 2, it is characterised in that the alcohol dehydrogenase comes from Escherichia coli or kerekou pneumonia primary
Bacterium.
7. the method for claim 1, it is characterised in that step (2) raw material containing fermentable sugars is molasses, sugarcane
Sugar, glucose, starch hydrolyzate, corn pulp, xylose, mannose or glycerine.
8. the method as described in claim 1-7 is any, it is characterised in that the fermentation condition of step (2) is, 28-35 DEG C, pH value
5-8, oxygen dissolving value 10%.
9. the method as described in claim 1-7 is any, it is characterised in that also including stream plus carbon source, Yi Jifa in fermentation process
Contain thiamine and vitamin B12 in ferment culture medium.
10. application of any described methods of claim 1-9 in 1,3- propanediols are improved.
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